Files in this item

Abstract

English

The first part of this thesis deals with the x-ray
crystal structure of the C2B domain of Rabphilin-3A,
which includes part of the linker region between the
C2A and C2B domain. The second part deals with the
x-ray crystal structure of the PP2C-like phosphatase
tPphA of Thermosynechococcus
elongatus BP-1. Both structures could be solved
after expression, purification and crystallisation of
the native and Se-Met protein via MAD
(multiple-wavelength anomalous dispersion). In both
cases crystals in two different space groups were
obtained which were compared to each other.C2 domains are intracellular protein moduls of about
130 amino acids. They contain a characteristic
structure motif of an eight-stranded antiparallel
β-sandwich and show high calcium ion affinity. They are
involved in exocytosis and in the regulation of the
neurotransmitter release in the synaptic vesicle cycle.
Rabphilin-3A contains a Rab binding domain and two C2
domains (C2A and C2B). The NMR-structure of the C2B
domain was already known. But since it could not
explain the high calcium ion affinity the cystal
structure of the C2B domain and part of the linker
region were examined in this thesis. The crystal
structure differs from the NMR-structure in the calcium
binding site and explains the high calcium affinity by
ideal coordination of the two calcium ions.Cyanobacteria are able to use nitrate, nitrite or
ammonia as a nitrogen source (nitrogen assimilation)
and molecular nitrogen for building biologically usable
nitrogen compounds (nitrogen fixation). Both nitrogen
assimilation and nitrogen fixation lead to the
formation of ammonia which is the central starting
point for the GS/GOGAT-cycle (glutamine
synthetase/glutamat-2-oxoglutarat amidotransferase).
This cycle is controlled via regulation of the
glutamine synthetase which is taken over from the PII
proteins. The PII proteins will be phosphorylated or
dephosphorylated depending on the carbon-nitrogen
balance of the cell to regulate the activity of the
glutamine synthetase. Dephosphorylation is done by a
PP2C-like phosphatase (PII phosphatase): tPphA. The
structure of tPphA was examined via x-ray
crystallography and compared with other PP2C-like
phosphatases to get an idea of the catalytic mechanism.
The high degree of similarity between tPphA and the
bacterial phosphatase indicates a similar mechanism of
the two phosphatases. The mechanism itself is yet
unexplained in either cases.